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adducts are formed in DNA by 1,2,3,4-diepoxybutane (metabolite of human carcinogen 1,3-butadiene). Results: hpols and carry out translesion synthesis, incorporating T, G, or A opposite the 1,N

Abstract1,2,3,4-Diepoxybutane (DEB) is a carcinogenic metabolite of 1,3-butadiene (BD), an important industrial and environmental chemical present in urban air and in cigarette smoke. DEB is considered the ultimate carcinogenic species of BD due to its potent genotoxicity and mutagenicity attributed to its ability to form DNA-DNA cross-links and exocyclic nucleoside adducts. Mutagenesis studies suggest that DEB adducts formed at adenine bases may be critically important, as it induces large numbers of A → T transversions. We have recently identified three types of exocyclic DEB-dA lesions: N 6 ,N 6 -(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (N 6 ,N 6 -DHB-dA), 1,N 6 -(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,N 6 -γ-HMHP-dA), and 1,N 6 -(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2′-deoxyadenosine (1,N 6 -α-HMHP-dA) (Seneviratne et al Chem. Res. Toxicol. 2010, 23, 118-133). In the present work, a post-synthetic methodology for preparing DNA oligomers containing stereo-and site-specific N 6 ,N 6 -DHB-dA and 1,N 6 -γ-HMHP-dA adducts was developed. DNA oligomers containing site specific 6-chloropurine were coupled with optically pure 1-amino-2-hydroxy-3,4-epoxybutanes to generate oligomers containing N 6 -(2-hydroxy-3,4-epoxybut-1-yl)adenine adducts, followed by their spontaneous cyclization to 1,N 6 -γ-HMHP-dA lesions. N 6 ,N 6 -DHB-dA containing strands were prepared analogously by coupling 6-chloropurine containing DNA with 3S,4S or 3R,4R pyrrolidine-3,4-diols. Oligodeoxynucleotide structures were confirmed by ESI-MS, exonuclease ladder sequencing, and HPLC-MS/MS of enzymatic digests. UV melting and CD spectroscopy studies of DNA duplexes containing N 6 ,N 6 -DHB-dA and 1,N 6 -γ-HMHP-dA revealed that both lesions lower the thermodynamic stability of DNA. Interestingly, structurally modified DNA duplexes were more thermodynamically stable when adenine residue was placed opposite 1,N 6 -γ-HMHP-dA instead of thymine, suggesting that these adducts may preferentially pair with dA.

Background 1,3-Butadiene (BD) is an important carcinogen in tobacco smoke that undergoes metabolic activation to DNA-reactive epoxides. These species can be detoxified via glutathione conjugation and excreted in urine as the corresponding N-acetylcysteine conjugates. We hypothesize that single nucleotide polymorphisms in BD-metabolizing genes may change the balance of BD bioactivation and detoxification in White, Japanese American, and African American smokers, potentially contributing to ethnic differences in lung cancer risk. Methods We measured the levels of BD metabolites, 1- and 2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene (MHBMA) and N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (DHBMA), in urine samples from a total of 1,072 White, Japanese American, and African American smokers and adjusted these values for body mass index, age, batch, and total nicotine equivalents. We also conducted a genome wide association study to identify genetic determinants of BD metabolism. Results We found that mean urinary MHBMA concentrations differed significantly by ethnicity (p = 4.0 × 10−25). African Americans excreted the highest levels of MHBMA followed by Whites and Japanese Americans. MHBMA levels were affected by GSTT1 gene copy number (p < 0.0001); conditional on GSTT1, no other polymorphisms showed a significant association. Urinary DHBMA levels also differed between ethnic groups (p = 3.3 ×10−4), but were not affected by GSTT1 copy number (p = 0.226). Conclusions GSTT1 gene deletion has a strong effect on urinary MHBMA levels, and therefore BD metabolism, in smokers. Impact Our results show that the order of MHBMA levels among ethnic groups is consistent with their respective lung cancer risk and can be partially explained by GSTT1 genotype.

Human carcinogen 1,3-butadiene (BD) undergoes metabolic activation to 3,4-epoxy-1-butene (EB), hydroxymethylvinyl ketone (HMVK), 3,4-epoxy-1,2-butanediol (EBD) and 1,2,3,4-diepoxybutane (DEB). Among these, DEB is by far the most genotoxic metabolite and is considered the ultimate carcinogenic species of BD. We have shown previously that BD-exposed laboratory mice form 8- to 10-fold more DEB-DNA adducts than rats exposed at the same conditions, which may be responsible for the enhanced sensitivity of mice to BD-mediated cancer. In the present study, we have identified 1,4-bis-(N-acetyl-L-cystein-S-yl)butane-2,3-diol (bis-BDMA) as a novel DEB-specific urinary biomarker. Isotope dilution high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was employed to quantify bis-BDMA and three other BD-mercapturic acids, 2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene/1-(N-acetyl-L-cystein-S-yl)-2-hydroxy-but-3-ene (MHBMA, from EB), 4-(N-acetyl-L-cystein-S-yl)-1,2-dihydroxybutane (DHBMA, from HMVK) and 4-(N-acetyl-L-cystein-S-yl)-1,2,3-trihydroxybutane (THBMA, from EBD), in urine of confirmed smokers, occupationally exposed workers and BD-exposed laboratory rats. Bis-BDMA was formed in a dose-dependent manner in urine of rats exposed to 0-200 p.p.m. BD by inhalation, although it was a minor metabolite (1%) as compared with DHBMA (47%) and THBMA (37%). In humans, DHBMA was the most abundant BD-mercapturic acid excreted (93%), followed by THBMA (5%) and MHBMA (2%), whereas no bis-BDMA was detected. These results reveal significant differences in metabolism of BD between rats and humans.

N 6-(2-Hydroxy-3-buten-1-yl)-2′-deoxyadenosine (N6-HB-dA I) and N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (N6,N6-DHB-dA) are exocyclic DNA adducts formed upon alkylation of the N6 position of adenine in DNA by epoxide metabolites of 1,3-butadiene (BD), a common industrial and environmental chemical classified as a human and animal carcinogen. Since the N6-H atom of adenine is required for Watson-Crick hydrogen bonding with thymine, N6-alkylation can prevent adenine from normal pairing with thymine, potentially compromising the accuracy of DNA replication. To evaluate the ability of BD-derived N6-alkyladenine lesions to induce mutations, synthetic oligodeoxynucleotides containing site-specific (S)-N6-HB-dA I and (R,R)-N6,N6-DHB-dA adducts were subjected to in vitro translesion synthesis in the presence of human DNA polymerases β, η, ι, and κ. While (S)-N6-HB-dA I was readily bypassed by all four enzymes, only polymerases η and κ were able to carry out DNA synthesis past (R,R)-N6,N6-DHB-dA. Steady-state kinetic analyses indicated that all four DNA polymerases preferentially incorporated the correct base (T) opposite (S)-N6-HB-dA I. In contrast, hPol β was completely blocked by (R,R)-N6,N6-DHB-dA, while hPol η and κ inserted A, G, C, or T opposite the adduct with similar frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed that while translesion synthesis past (S)-N6-HB-dA I was mostly error-free, replication of DNA containing (R,R)-N6,N6-DHB-dA induced significant numbers of A, C, and G insertions and small deletions. These results indicate that singly substituted (S)-N6-HB-dA I lesions are not miscoding, but exocyclic (R,R)-N6,N6-DHB-dA adducts are strongly mispairing, probably due to their inability to form stable Watson-Crick pairs with dT.

Background We hypothesize that the differences in lung cancer risk in Native Hawaiians (NH), whites and Japanese Americans (JA), may in part be due to variation in the metabolism of 1,3-butadiene (BD), one of the most abundant carcinogens in cigarette smoke. Methods We measured two biomarkers of BD exposure, monohydroxybutyl mercapturic acid (MHBMA) and dihydroxybutyl mercapturic acid (DHBMA) in overnight urine samples among 585 NH, JA and white smokers in Hawaii. These values were normalized to creatinine levels. Ethnic-specific geometric means were compared adjusting for age at urine collection, sex, body mass index and nicotine equivalents (a marker of total nicotine uptake). Results We found that mean urinary MHBMA differed by race/ethnicity (p=0.0002). The values were highest in whites and lowest in JA. This difference was only observed in individuals with the GSTT1-null genotype (p=0.0001). No difference across race/ethnicity was found among those with at least one copy of the GSTT1 gene (p≥0.72). Mean urinary DHBMA did not differ across racial/ethnic groups. Conclusions The difference in urinary MHBMA excretion levels from cigarette smoking across three ethnic groups is in part explained by the GSTT1 genotype. Mean urinary MHBMA levels are higher in whites among GSTT1-null smokers. Impact The overall higher excretion levels of MHBMA in whites and lower levels of MHBMA in JA are consistent with the higher lung cancer risk in the former. However, the excretion levels of MHBMA in NH are not consistent with and, thus, unlikely to explain, their high risk of lung cancer.

Antibody drug conjugates (ADCs) can undergo in vivo biotransformation (e.g., payload metabolism, deconjugation) leading to reduced or complete loss of activity. The location/site of conjugation of payload-linker can have an effect on ADC stability and hence needs to be carefully optimized. Affinity capture LC–MS of intact ADCs or ADC subfragments has been extensively used to evaluate ADC biotransformation. However, the current methods have certain limitations such as the requirement of specific capture reagents, limited mass resolution of low mass change metabolites, low sensitivity, and use of capillary or nanoflow LC–MS. To address these challenges, we developed a generic affinity capture LC–MS assay that can be utilized to evaluate the biotransformation of any site-specific ADC independent of antibody type and site of conjugation (Fab and Fc) in preclinical studies. The method involves a combination of some or all of these steps: (1) “mono capture” or “dual capture” of ADCs from serum with streptavidin magnetic beads coated with a generic biotinylated antihuman capture reagent, (2) “on-bead” digestion with IdeS and/or PNGase F, and (3) reduction of interchain disulfide bonds to generate ∼25 kDa ADC subfragments, which are finally analyzed by LC–HRMS on a TOF mass spectrometer. The advantages of this method are that it can be performed using commercially available generic reagents and requires sample preparation time of less than 7 h. Furthermore, by reducing the size of intact ADC (∼150 kDa) to subfragments (∼25 kDa), the identification of conjugated payload and its metabolites can be achieved with excellent sensitivity and resolution (hydrolysis and other small mass change metabolites). This method was successfully applied to evaluate the in vitro and in vivo biotransformation of ADCs conjugated at different sites (LC, HC-Fab, and HC-Fc) with various classes of payload-linkers.